1. Field of the Invention
The present invention relates generally to transgenic plants. More specifically, it relates to methods and compositions for transgene expression using the maize A3 promoter.
2. Description of the Related Art
An important aspect in the production of genetically engineered crops is obtaining sufficient levels of transgene expression in the appropriate plant tissues. In this respect, the selection of promoters for directing expression of a given transgene is crucial. Promoters which are useful for plant transgene expression include those that are inducible, viral, synthetic, constitutive as described (Poszkowski et al., 1989; Odell et al, 1985), temporally regulated, spatially regulated, and spatio-temporally regulated (Chau et al, 1989).
A number of plant promoters have been described with various expression characteristics. Examples of some constitutive promoters which have been described include the CaMV 35S (Odell et al., 1985), CaMV 19S (Lawton et al., 1987), nos (Ebert et al, 1987), rice actin 1 (Wang et al., 1992; U.S. Pat. No. 5,641,876), and Adh (Walker et al., 1987).
Examples of tissue specific promoters which have been described include the lectin (Vodkin et al., 1983; Lindstrom et al., 1990), corn alcohol dehydrogenase 1 (Vogel et al., 1989; Dennis et al., 1984), corn light harvesting complex (Simpson, 1986; Bansal et al., 1992), corn heat shock protein (Odell et al., 1985; Rochester et al, 1986), pea small subunit RuBP carboxylase (Poulsen et al, 1986; Cashmore et al, 1983), Ti plasmid mannopine synthase (Langridge et al., 1989), Ti plasmid nopaline synthase (Langridge et al., 1989), petunia chalcone isomerase (Van Tunen et al, 1988), bean glycine rich protein 1 (Keller et al., 1989), truncated CaMV 35s (Odell et al, 1985), potato patatin (Wenzler et al., 1989), root cell (Conkling et al., 1990), maize zein (Reina et al., 1990; Kriz et al., 1987; Wandelt and Feix, 1989; Langridge and Feix, 1983; Reina et al., 1990), globulin-1 (Belanger and Kriz, 1991), xcex1-tubulin, cab (Sullivan et al., 1989), PEPCase (Hudspeth and Grula, 1989), sucrose synthase (Yang and Russell, 1990), R gene complex-associated promoters (Chandler et al, 1989), and chalcone synthase promoters (Franken et al., 1991).
Inducible promoters which have been described include ABA- and turgor-inducible promoters, the promoter of the auxin-binding protein gene (Schwob et al., 1993), the UDP glucose flavonoid glycosyl-transferase gene promoter (Ralston et al., 1988); the MPI proteinase inhibitor promoter (Cordero et al., 1994), and the glyceraldehyde-3-phosphate dehydrogenase gene promoter (Kohler et al., 1995; Quigley et al., 1989; Martinez et al., 1989).
A class of genes which are expressed in an-inducible manner are glycine rich proteins. Expression of glycine rich proteins is induced by the plant hormone abscibic acid (ABA). Genes encoding glycine rich proteins have been described, for example, from maize (Didierjean et al., 1992; Gomez et al., 1988; Baysdorfer, Genbank Accession No. AF034945) sorghum (Cretin and Puigdomenech, 1990), and rice (Lee et al., Genbank Accession No. AF009411).
In addition to the use of a particular promoter, expression of transgenes can be influenced by other types of elements. In particular, introns have demonstrated the potential for enhancing transgene expression. For example, Callis et al. (1987) described an intron from the corn alcohol dehydrogenase gene which is capable of enhancing the expression of transgenes in transgenic plant cells. Similarly, Vasil et al. (1989) described an intron from the corn sucrose synthase gene having similar enhancing activity. The rice actin 1 intron, in particular, has found wide use in the enhancement of transgene expression in a number of different transgenic crops (McElroy et al., 1991). This 5xe2x80x2 intron was identified from the first coding exon of the rice actin 1 sequence (McElroy et al., 1990a). Plant actin is encoded by a gene family present in all plant species studied to date (Meagher, 1991). In rice, there are at least eight actin-like sequences per haploid genome. Four of the rice actin coding sequences (rice actin 1, 2, 3 and 7) have been isolated and shown to differ from each other in the tissue and stage-specific abundance of their respective transcripts (Reece. 1988; McElroy et al., 1990a; Reece et al, 1990; U.S. Pat. No. 5,641,876; Genbank Accession numbers X15865, X15864, X15862, and X15863, respectively).
While the above studies have provided a number of useful tools for the generation of transgenic plants, there is still a great need in the art for novel promoter and enhancer sequences with beneficial expression characteristics. In particular, there is a need in the art for promoter-enhancer combinations which are capable of directing high-level expression of exogenous genes in transgenic crop plants. Many previously identified regulatory sequences fail to provide the levels of expression required to fully realize the benefits potentially conferred by expression of selected genes in transgenic plants. Additionally, many regulatory regions fail to demonstrate suitable or desirable expression profiles for transgene expression. There is, therefore, a great need in the art for the identification of novel sequences which can be used for the high-level expression of selected transgenes in economically important crop plants.
Thus, in accordance with the present invention, there is provided, in a first embodiment, an isolated nucleic acid comprising a maize A3 promoter. The maize A3 promoter is, in one embodiment, isolatable from the nucleic acid sequence of SEQ ID NO:4. For example, the promoter may comprise from about 100 to about 1294 contiguous nucleotides, from about 150 to about 1294 contiguous nucleotides, from about 250 to about 1294 contiguous nucleotides, from about 400 to about 1294 contiguous nucleotides, from about 750 to about 1294 contiguous nucleotides, or from about 1000 to about 1294 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:4. Alternatively, the maize A3 promoter can comprise the entire nucleic acid sequence of SEQ ID NO:4.
The isolated nucleic acid also may comprise a rice actin 2 intron. The rice actin 2 intron is, in one embodiment, be isolatable from the nucleic acid sequence of SEQ ID NO:2. For example, the intron may comprise from about 40 to about 1763 contiguous nucleotides, from about 80 to about 1763 contiguous nucleotides, from about 150 to about 1763 contiguous nucleotides, from about 300 to about 1763 contiguous nucleotides, or from about 600 to about 1763 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:2. Alternatively, the intron may include the entire nucleic acid sequence of SEQ ID NO:2.
In another embodiment, the invention comprises an expression vector comprising a maize A3 promoter operably linked to a selected DNA sequence. The selected DNA may be a coding region for insect resistance protein, a bacterial disease resistance protein, a fungal disease resistance protein, a viral disease resistance protein, a nematode disease resistance protein, a herbicide resistance protein, a protein affecting grain composition or quality, a nutrient utilization protein, a mycotoxin reduction protein, a male sterility protein, a selectable marker protein, a screenable marker protein, a negative selectable marker protein, an environment or stress resistance protein, or a protein affecting plant agronomic characteristics.
The selectable marker protein may be a protein selected from the group consisting of phosphinothricin acetyltransferase, glyphosate resistant EPSPS, aminoglycoside phosphotransferase, hygromycin phosphotransferase, neomycin phosphotransferase, dalapon dehalogenase, bromoxynil resistant nitrilase and anthranilate synthase.
An expression vector may have the selected coding region operably linked to a terminator. The expression vector may further be defined as a plasmid vector. The expression vector may further comprise a genetic element that enhances the expression of said selected gene in a transgenic plant transformed with said expression vector. The genetic element comprises an actin 1 intron or an actin 2 intron. The expression vector may further comprise a transit peptide coding sequence.
The transit peptide may be selected from the group consisting of chlorophyll a/b binding protein transit peptide, small subunit of ribulose bisphosphate carboxylase transit peptide, EPSPS transit peptide and dihydrodipocolinic acid synthase transit peptide.
In yet another embodiment, there is provided a fertile transgenic plant stably transformed with a selected DNA comprising a maize A3 promoter. The maize A3 promoter may be isolatable from the nucleic acid sequence of SEQ ID NO:4, as described above. The selected DNA may further comprise a selected coding region operably linked to said maize A3 promoter. The selected coding region may encode an insect resistance protein, a bacterial disease resistance protein, a fungal disease resistance protein, a viral disease resistance protein, a nematode disease resistance protein, a herbicide resistance protein, a protein affecting grain composition or quality, a nutrient utilization protein, an environment or stress resistance protein, a mycotoxin reduction protein, a male sterility protein, a selectable marker protein, a screenable marker protein, a negative selectable marker protein, or a protein affecting plant agronomic characteristics.
The fertile transgenic plant may also comprise a selected DNA comprising a transit peptide coding sequence. The fertile transgenic plant may further be a monocotyledonous plant, for example, a monocotyledonous plant selected from the group consisting of wheat, maize, rye, rice, oat, barley, turfgrass, sorghum, millet and sugarcane. The fertile transgenic plant also may be a dicotyledonous plant, for example, a dicotyledonous plant is selected from the group consisting of tobacco, tomato, potato, soybean, cotton, canola, sunflower and alfalfa. The fertile transgenic plant may be further defined as an Ro transgenic plant, or seed thereof. The fertile transgenic plant also may be further defined as a progeny plant of any generation of an Ro transgenic plant, wherein said Ro transgenic plant comprises said selected DNA, and seed thereof.
In still yet another embodiment, there is provided a crossed fertile transgenic plant prepared according to the method comprising the steps of (i) obtaining a fertile transgenic plant comprising a selected DNA comprising a maize A3 promoter; (ii) crossing said fertile transgenic plant with itself or with a second plant lacking said selected DNA to prepare the seed of a crossed fertile transgenic plant, wherein said seed comprises said selected DNA; and (iii) planting said seed to obtain a crossed fertile transgenic plant. Seed of the crossed fertile transgenic also is encompassed by the present invention.
The crossed fertile transgenic plant may be a monocotyledonous plant or a dicotyledonous plant, as described above. The selected DNA may be inherited through a female parent or a male parent. The crossed fertile transgenic plant may be an inbred plant or a hybrid. The maize A3 promoter may be isolatable from the nucleic acid sequence of SEQ ID NO:4.
The crossed fertile transgenic plant may have a selected DNA may further comprise a selected coding region operably linked to said maize A3 promoter. The selected coding region may encode a protein selected from the group consisting of an insect resistance protein, a bacterial disease resistance protein, a fungal disease resistance protein, a viral disease resistance protein, a nematode disease resistance protein, a herbicide resistance protein, a protein affecting grain composition or quality, a nutrient utilization protein, a mycotoxin reduction protein, a male sterility protein, a selectable marker protein, a screenable marker protein, a negative selectable marker protein, a protein affecting plant agronomic characteristics, and an environment or stress resistance protein.
The crossed fertile transgenic may also have a selected DNA comprising a genetic element which enhances the expression of said selected DNA in said crossed fertile transgenic plant. The genetic element may be the rice actin 1 intron or rice actin 2 intron.
In still a further embodiment, there is provided a method of expressing a selected DNA in a transgenic plant comprising the steps of (i) obtaining a construct comprising a selected DNA operably linked to a maize A3 promoter; (ii) transforming a recipient plant cell with said construct; and (iii) regenerating a transgenic plant expressing said selected gene from said recipient plant cell. The transforming may comprise microprojectile bombardment. The plant cell may be a monocotyledonous plant or a dicotyledonous plant.
The selected DNA may be a coding region encoding an insect resistance protein, a bacterial disease resistance protein, a fungal disease resistance protein, a viral disease resistance protein, a nematode disease resistance protein, a herbicide resistance protein, a protein affecting grain composition or quality, a nutrient utilization protein, a mycotoxin reduction protein, a male sterility protein, a selectable marker protein, a screenable marker protein, a negative selectable marker protein, a protein affecting plant agronomic characteristics, and an environment or stress resistance protein. The construct may further comprise a genetic element which enhances the expression of said selected DNA in said transgenic plant, for example the rice actin 1 intron and rice actin 2 intron.
In still yet a further embodiment, there is provided a method of plant breeding comprising the steps of (i) obtaining a transgenic plant comprising a selected DNA comprising a maize A3 promoter; and (ii) crossing said transgenic plant with itself or a second plant. The plant may be a monocotyledonous or dicotyledonous plant. The maize A3 promoter may be isolatable from the nucleic acid sequence of SEQ ID NO:4. The transgenic plant may be crossed with said second plant, for example, an inbred plant.
The method may further comprise the steps of (iii) collecting seeds resulting from said crossing; (iv) growing said seeds to produce progeny plants; (v) identifying a progeny plant comprising said selected DNA; and (vi) crossing said progeny plant with itself or a third plant. The progeny plant may inherit the selected DNA through a female parent or a male parent. The second plant and said third plant may be of the same genotype. The second and third plants may be inbred plants. The selected DNA may further encode a protein selected from the group consisting of an insect resistance protein, a bacterial disease resistance protein, a fungal disease resistance protein, a viral disease resistance protein, a nematode disease resistance protein, a herbicide resistance protein, a protein affecting grain composition or quality, a nutrient utilization protein, a mycotoxin reduction protein, an environment or stress resistance protein, a male sterility protein, a selectable marker protein, a screenable marker protein, a negative selectable marker protein, and a protein affecting plant agronomic characteristics. The selected DNA may further comprise a genetic element which enhances the expression of said gene in said transgenic plant, for example, the rice actin 1 intron and rice actin 2 intron.
All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.